Variants of the NK1 fragment of hepatocyte growth factor/scatter factor (HGF/SF) and their use

ABSTRACT

Variants of the NK1 fragment of the polypeptide growth factor HGF/SF which act as agonists of the MET receptor and their use are disclosed. The agonists comprise at least one substitution at positions equivalent to 132, 134, 170 and 181 of full length HGF/SF (SEQ ID NO:2) and these substitutions provide variants which show scatter factor activity and induce DNA synthesis. In vivo, the variants provide protection from liver damage in a model of acute liver failure.

FIELD OF THE INVENTION

The present invention relates to variants of the NK1 fragment of thepolypeptide growth factor HGF/SF which act as agonists of the METreceptor, and to the use of NK1 and its variants in methods oftreatment.

BACKGROUND TO THE INVENTION

The polypeptide growth factor hepatocyte growth factor/scatter factor(HGF/SF) (Gherardi et al., 1989; Miyazawa et al., 1989; Nakamura et al.,1989; Stoker et al., 1987) and its receptor MET, the product of thec-MET protoncogene (Bottaro et al., 1991), play essential roles in thedevelopment of epithelial organs such as the placenta and liver (Schmidtet al., 1995; Uehara et al., 1995) and in the migration of myogenicprecursor cells (Bladt et al., 1995) and motor neurons (Caton et al.,2000; Ebens et al., 1996).

HGF/SF and MET are also involved in the spreading of a variety ofepithelial tumours as a result of MET chromosomal rearrangements (Yu etal., 2000), somatic and/or germline mutations in the MET kinase (Schmidtet al., 1997) or, more often, over expression in tumour cells of anunrearranged and unmutated MET gene (reviewed in Jeffers et al., 1996).

HGF/SF has a unique domain structure that resembles that of the bloodproteinase precursor plasminogen and consists of six domains: anN-terminal (N) domain, homologous to plasminogen activation peptide,four copies of the kringle (K) domain and a catalytically inactiveserine proteinase domain (Donate et al., 1994). Two products ofalternative splicing of the primary HGF/SF transcript encode NK1, afragment containing the N and the first K domain, K1, (Cioce et al.,1996)., and NK2, a fragment containing the N, K1 and second kringle, K2,domains (Chan et al., 1991; Hartmann et al., 1992; Miyazawa et al.,1991). Both NK1 (Lokker and Godowski, 1993) and NK2 (Chan et al., 1991)were initially characterized as MET antagonists, although experiments intransgenic mice have subsequently indicated that NK1 behaves in vivo asa bona fide receptor agonist (Jakubczak et al., 1998).

There is an important difference in the mechanism of receptor bindingand activation by HGF/SF and NK1. HGF/SF is fully active in cellslacking heparan sulphate, while NK1 is only active in cells that displayheparan sulphate or in the presence of soluble heparin (Schwall et al.,1996). Thus NK1, but not HGF/SF, resembles FGF (Rapraeger et al., 1991;Yayon et al., 1991) in terms of a requirement for heparan sulphate forreceptor binding and/or activation.

Early domain deletion experiments indicated that the N domain isimportant for heparin binding (Mizuno et al., 1994) and site-directedmutagenesis identified residues in this domain essential for binding(Hartmann et al., 1998). Thus reverse-charge mutation of R73 and R76decreased the affinity of HGF/SF for heparin by more than 50 fold(Hartmann et al., 1998). A role for several other positively-chargedresidues, such as K58, K60 and K62, was suggested from the solutionstructure of the N domain, as these residues are clustered in closeproximity of R73 and R76 (Zhou et al., 1998), and recent NMR experimentshave provided experimental support for an involvement of K60, K62, R73,R76, R78 and several other residues in heparin binding to the N domain(Zhou et al., 1999).

Despite this progress, the mechanism through which heparin and heparansulphate confer agonistic activity to NK1 remains incompletelyunderstood. NK1 crystallizes as a dimer in the absence of heparin(Chirgadze et al., 1999; Ultsch et al., 1998), and the features of thisdimer suggested that it could represent the biologically active form ofNK1 (Chirgadze et al., 1999). No experimental evidence, however,supports this interpretation as yet.

Sequence Listing

SEQ ID NO:1 represents amino acids 28 to 210 of the human HGF/SFprotein. Residues 32–206 of HGF/SF are the wild type NK1 fragment. Wehave used short N- and C-terminal extensions as a matter of experimentalconvenience to optimise expression in yeast.

SEQ ID NO:2 is the full length HGF/SF sequence, of which residues 1–31are the leader sequence and 32–206 the NK1 fragment.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows DNA synthesis assays using MK cells. The cells werecultured to confluence in keratinocyte serum-free medium and transferredin basal medium for 24 hours before incubation with ³H-thymidine andHGF/SF or NK1 proteins at the concentrations (mol/L) indicated in theFigure (x-axis). DNA synthesis was measured as TCA-insolubleradioactivity; the Y axis shows ³H-thymidine incorporation,cpm×10³/well. The HP11 mutant is inactive and the HP12 shows muchreduced activity compared to wt-NK1. In contrast the 1K1 mutant is moreactive than wt-NK1 and full length HGF/SF.

FIG. 2 shows the survival rates of Balb/c mice after administration of alethal dose of N-acetyl-p-aminophenol followed by treatment with NK1 anda peptide of the invention.

FIG. 3 shows the survival rates of Balb/c mice after administration of alethal dose of alpha-amanitin.

DISCLOSURE OF THE INVENTION

We have determined two X-ray crystal structures of NK1-heparin complexesthat define the heparin-binding site of NK1. Our analysis of thesestructures confirms that contacts between heparin and residues in theN-domain occur. Surprisingly though, our analysis also identifies anumber of critical heparin contacts with four positively chargedresidues in the K1 domain. More surprisingly, we have furtherdemonstrated that heparin binding to these positively charged residuesin the K1 domain inhibits activity, and that mutagenesis of the residuesprovides NK1 variants with higher than wild-type activity. Such variantsare useful for the production of agonists for the promotion of cellgrowth, particularly for angiogenesis, and the treatment ofcardiovascular, hepatic, musculoskeletal and neuronal diseases.

Thus, the present invention provides a polypeptide variant of SEQ IDNO:1, said variant having the sequence of SEQ ID NO:1 apart from asubstitution or deletion of least one of positions corresponding to 132,134, 170 and 181 of HGF/SF. For ease of reference, positions of SEQ IDNO:1 are defined in relation to full length HGF/SF (SEQ ID NO:2) unlessstated to the contrary. The variant is one which retains the ability toexhibit heparin-dependent dimerization in solution and to act as anagonist against the MET receptor.

The invention also provides a polypeptide which is fragment of thepolypeptide variant of the invention, said fragment retaining the132–181 region and further retaining the ability to exhibitheparin-dependent dimerization in solution and to act as an agonistagainst the MET receptor.

The invention further provides a composition comprising a polypeptide ofthe invention together with a pharmaceutically acceptable diluent orcarrier.

The invention further provides a method for stimulating the growth of acell which expresses the MET receptor, said method comprising bringing apolypeptide of the invention into contact with said cell. The cell maybe in vitro or in vivo.

The invention further provides a method of treatment of a patient havinga disease condition which requires stimulation of cell growth, saidmethod comprising administering to a patient an effective amount of apolypeptide of the invention.

The invention also provides a polypeptide of the invention for use in amethod of treatment of the human or animal body.

The invention further provides a polynucleotide coding for a polypeptideof the invention, as well as vectors carrying said polynucleotide,including expression vectors wherein the polynucleotide is operablylinked to a promoter.

The invention further provides a host cell carrying a vector of theinvention, and methods for the production of a polypeptide of theinvention which comprises culturing the host cells under conditionssuitable for the expression of the polynucleotide carried by the vector,and recovering the polypeptide from the cell or culture.

A major effort is underway for developing MET antagonists and agonistsfor therapy. MET antagonists are expected to find applications in avariety of epithelial tumours over-expressing MET, while receptoragonists may be valuable in liver regeneration, the repair of skinwounds and therapeutic angiogenesis. The structural and mutagenesis dataprovided by the present invention enables the generation of potent METagonists.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides

Polypeptides of the invention are those in which one of positions 132,134, 170 and 181 are substituted with any other amino acid. It ispreferred, however, that the substitutions are those which result in achange of charge. Preferred substitutions thus include reverse chargesubstitutions of aspartic acid and glutamic acid.

Two or more of the positions may be substituted simultaneously. Wheretwo substitutions are made, in a preferred aspect the two are either 132and 134 or 170 and 181. Three substitutions may also be made or all fourpositions may be substituted. Where more than one position issubstituted the substitutions may be the same or different.

Polypeptides of the invention may be prepared in isolated form. Isolatedpolypeptides of the invention will be those as defined above in isolatedform, free or substantially free of material with which it is naturallyassociated such as other polypeptides with which it is found in thecell. The polypeptides may of course be formulated with diluents oradjuvants and still for practical purposes be isolated. The polypeptidesmay be glycosylated, either naturally or by systems of heterologouseukaryotic cells, or they may be (for example if produced by expressionin a prokaryotic cell) unglycosylated.

A polypeptide of the invention may also be in a substantially purifiedform, in which case it will generally comprise the polypeptide in apreparation in which more than 90%, e.g. 95%, 98% or 99% of thepolypeptide in the preparation is a polypeptide of the invention.

Polypeptides of the invention may be modified for example by theaddition of histidine residues to assist their purification or by theaddition of a signal sequence to promote their secretion from a cell.

SEQ ID NO: 1 represents the wild-type human form of NK1 (together withshort N- and C-terminal extension). However, those of skill in the artwill appreciate that in addition to the specific substitutions of theinvention which result in increased activity, other positions of thewild-type molecule may be varied to a small degree without significantlyaffecting the overall function or structure of the polypeptide. Forexample, conservative substitutions can be made to many parts ofproteins with no discernable impact on the structure or function of thatprotein. Those of skill in the art will appreciate that a small number,for example from 1–20, e.g. 2, 3, 4 or 5–10 other amino acidsubstitutions mainly made to NK1 and provided substitutions do notsignificantly alter the activity of polypeptides of the invention suchvariants are still regarded as NK1 polypeptides.

Fragments of the polypeptide variants of the invention which retain theregion 132–181 also form a further part of the invention. Such fragmentsmay be from 70 to 190 amino acids in size, for example from 100 to 180in size. An example of such a fragment is provided herein as the NK1fragment of amino acids 32–206. Another fragment is one which includesat least 70 contiguous amino acids of the Kringle 1 domain, which isfound at 128–206. Preferably the fragment contains this domain in itsentirety.

Methods to determine whether a polypeptide of the invention retains theability to exhibit heparin-dependent dimerization in solution are setout in the accompanying examples. As indicated, the polypeptide may beincubated in with an equimolar concentration of heparin in a 300 mM NaClbuffer and analysed by gel filtration or western blotting.

Likewise, the ability to act as an agonist of the MET receptor may bedetermined in accordance with the accompanying examples. This mayinvolve incubating the polypeptide with murine keratinocyte cells (e.g.MK cells) at a concentration of 10⁻¹⁰ M or higher (e.g. 10⁻¹⁰ to 10⁻⁸ M,and determining whether there is an increase in DNA synthesis in thecells.

A polypeptide according to the present invention may be isolated and/orpurified (e.g. using an antibody) for instance after production byexpression from encoding nucleic acid (for which see below).Polypeptides according to the present invention may also be generatedwholly or partly by chemical synthesis, for example in a step-wisemanner. The isolated and/or purified polypeptide may be used informulation of a composition, which may include at least one additionalcomponent, for example a pharmaceutical composition including apharmaceutically acceptable excipient, vehicle or carrier. A compositionincluding a polypeptide according to the invention may be used inprophylactic and/or therapeutic treatment as discussed below.

A polypeptide of the invention may be labelled with a revealing label.The revealing label may be any suitable label which allows thepolypeptide to be detected. Suitable labels include radioisotopes, e.g.¹²⁵I, enzymes, antibodies, polynucleotides and linkers such as biotin.Labelled polypeptides of the invention may be used in diagnosticprocedures such as immunoassays in order to determine the amount of apolypeptide of the invention in a sample.

Polypeptides and compositions thereof according to the invention may beused in methods of treatment. Such treatment will be directed topromoting the growth of cells in the human body which express the METreceptor. Such therapy will be useful for the promotion of angiogenesisand thus will be useful for the treatment of chronic skin wounds,chronic liver and kidney disease, degenerative musculoskeletelal andneuronal diseases and cardiovascular disease.

A particular use of the polypeptides of the invention is in thetreatment or prevention of liver damage caused by intoxication byN-acetyl-p-aminophenol (known commercially as paracetamol oracetaminophen). We have found that administration of a polypeptide ofthe invention in a mouse model substantially increases survival rates ofmice following administration of a lethal dose of paracetamol. Someprotection is also provided by the NK1 peptide itself. Thus in thisaspect of the invention, there is also provided the use of an NK1peptide for the above-mentioned treatment.

Thus the invention provides a method of treatment or prevention of liverdamage in a subject who has ingested N-acetyl-p-aminophenol, the methodcomprising administering to the subject an effective amount of apolypeptide of the invention or an NK1 polypeptide.

The invention also provides a polypeptide of the invention or an NK1polypeptide for use in a method of therapy of a human or animal subject,particularly for treatment or prevention of liver damage in a subjectwho has ingested N-acetyl-p-aminophenol.

We have also demonstrated that the NK1 peptide as well as the 1K1polypeptide is effective in treating acute liver failure caused byα-amanitin, which is a potent specific inhibitor of RNA polymerase II.Thus the NK1 peptide as well as the peptides of the invention as definedherein may be used generally in a method of treatment of liver disease,particularly disease conditions associated with liver failure. Suchconditions include not only toxicity caused by N-acetyl-aminophenonl,but also include other drug-induced and other causes of liver failure,or disease.

Thus these peptides may be used to treat or prevent acute liver failureor disease induced by toxins, including a toxin selected from mushroompoisoning (e.g. Amanita phalloides), arsenic, carbon tetrachloride (orother chlorinated hydrocarbons), copper, ethanol, iron, methotrexate andphosphorus.

The invention may further be used to treat or prevent liver failure ordisease caused by other means, including conditions selected from viralinfection (such as by infection with a hepatitis virus, e.g. HAV, HBV orHCV), or other acute viral hepatitis, autoimmune chronic hepatitis,acute fatty liver of pregnancy, Budd-Chiari syndrome and veno-occlusivedisease, hyperthermia, hypoxia, malignant infiltration, Reye's syndrome,sepsis, Wilson's disease and in transplant rejection.

Polypeptides may be administered in any suitable form, for example in apharmaceutical composition such as water, saline, dextrose, glycerol,ethanol and the like. Compositions may be formulated for injection, forexample for direct injection to the site of intended treatment orintravenous injection.

Suitable doses of polypeptides will ultimately be at the discretion ofthe physician taking account of the nature of the condition to betreated and the condition of the patient. In general, dosage ranges willbe 1 μg to 1 mg per kg body weight. The polypeptides may be administeredby any suitable route, e.g. by i.v. or i.p injection, or directly to thesite of treatment.

By “treatment” it will be understood that this refers to anyadministration of a polypeptide intended to alleviate the severity of adisease being treated, to provide relief from the symptoms of thedisease or to prevent or slow down the development of the disease in anindividual with a disease condition or at risk of developing the diseasecondition.

Polynucleotides.

A polynucleotide of the invention is one which encodes a polypeptide ofthe invention as defined above. This includes DNA and RNApolynucleotides. A polynucleotide of the invention may be single ordouble stranded.

Generally, a polynucleotide according to the present invention isprovided as an isolate, in isolated and/or purified form, or free orsubstantially free of material with which it is naturally associated,such as free or substantially free of nucleic acid flanking the gene inthe human genome, except possibly one or more regulatory sequence(s) forexpression.

Sequences encoding all or part of the polypeptides of the inventionand/or its regulatory elements can be readily prepared by the skilledperson using the information and references contained herein andtechniques known in the art (for example, see Sambrook, Fritsch andManiatis, “Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989, and Ausubel et al, Short Protocols in MolecularBiology, John Wiley and Sons, 1992). These techniques include the use ofsite directed mutagenesis of nucleic acid encoding NK1, as described inthe accompanying examples.

Vectors.

Polynucleotides of the invention can be incorporated into a recombinantreplicable vector. The vector may be used to replicate the nucleic acidin a compatible host cell. Thus in a further embodiment, the inventionprovides a method of making polynucleotides of the invention byintroducing a polynucleotide of the invention into a replicable vector,introducing the vector into a compatible host cell and growing the hostcell under conditions which bring about replication of the vector. Thevector may be recovered from the host cell. Suitable host cells aredescribed below in connection with expression vectors.

Expression Vectors.

Preferably, a polynucleotide of the invention in a vector is operablylinked to a control sequence which is capable of providing for theexpression of the coding sequence by the host cell, i.e. the vector isan expression vector.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under condition compatible with the controlsequences.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.'phage phagemid or baculoviral, cosmids, YACs, BACs, or PACs asappropriate. Vectors include gene therapy vectors, for example vectorsbased on adenovirus, adeno-associated virus, retrovirus (such as HIV orMLV) or alpha virus vectors.

The vectors may be provided with an origin of replication, optionally apromoter for the expression of the said polynucleotide and optionally aregulator of the promoter. The vectors may contain one or moreselectable marker genes, for example an ampicillin resistance gene inthe case of a bacterial plasmid or a neomycin resistance gene for amammalian vector. Vectors may be used in vitro, for example for theproduction of RNA or used to transfect or transform a host cell. Thevector may also be adapted to be used in vivo, for example in methods ofgene therapy. Systems for cloning and expression of a polypeptide in avariety of different host cells are well known. Suitable host cellsinclude bacteria, eukaryotic cells such as mammalian and yeast, andbaculovirus systems. Mammalian cell lines available in the art forexpression of a heterologous polypeptide include Chinese hamster ovarycells, HeLa cells, baby hamster kidney cells, COS cells and many others.

Promoters and other expression regulation signals may be selected to becompatible with the host cell for which the expression vector isdesigned. For example, yeast promoters include S. cerevisiae GAL4 andADH promoters, S. pombe nmt1 and adh promoter. Mammalian promotersinclude the metallothionein promoter which is can be included inresponse to heavy metals such as cadmium. Viral promoters such as theSV40 large T antigen promoter or adenovirus promoters may also be used.All these promoters are readily available in the art.

The vectors may include other sequences such as promoters or enhancersto drive the expression of the inserted nucleic acid, nucleic acidsequences so that the polypeptide is produced as a fusion and/or nucleicacid encoding secretion signals so that the polypeptide produced in thehost cell is secreted from the cell.

Vectors for production of polypeptides of the invention of for use ingene therapy include vectors which carry a mini-gene sequence of theinvention.

Vectors may be introduced into a suitable host cell as described aboveto provide for expression of a polypeptide of the invention. Thus, in afurther aspect the invention provides a process for preparingpolypeptides according to the invention which comprises cultivating ahost cell carrying an expression vector as described above underconditions to provide for expression by the vector of a coding sequenceencoding the polypeptides, and recovering the expressed polypeptides.Polypeptides may also be expressed in in-vitro systems, such asreticulocyte lysate.

A further embodiment of the invention provides host cells carrying thevectors for the replication and expression of polynucleotides of theinvention. The cells will be chosen to be compatible with the saidvector and may for example be bacterial, yeast, insect or mammalian.

The introduction of vectors into a host cell may be followed by causingor allowing expression from the nucleic acid, e.g. by culturing hostcells (which may include cells actually transformed although more likelythe cells will be descendants of the transformed cells) under conditionsfor expression of the gene, so that the encoded polypeptide is produced.If the polypeptide is expressed coupled to an appropriate signal leaderpeptide it may be secreted from the cell into the culture medium.Following production by expression, a polypeptide may be isolated and/orpurified from the host cell and/or culture medium, as the case may be,and subsequently used as desired, e.g. in the formulation of acomposition which may include one or more additional components, such asa pharmaceutical composition which includes one or more pharmaceuticallyacceptable excipients, vehicles or carriers (e.g. see below).

A further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. The polynucleotides andvectors of the invention may be integrated into the genome (e.g.chromosome) of the host cell. Integration may be promoted by inclusionof sequences which promote recombination with the genome, in accordancewith standard techniques. The nucleic acid may be on anextra-chromosomal vector within the cell.

In the accompanying examples we show that wt-NK1 behaved as a partialagonist, as expected from the prior art. It produced full dispersion ofMDCK colonies and stimulation of DNA synthesis in MK cells (FIG. 1).Interestingly, maximal stimulation of DNA synthesis by wt-NK1 occurredat concentrations as low as 10⁻¹⁰ M, a concentration much lower thanthose required in other studies see for example (Schwall et al., 1996).The higher potency of NK1 observed in our studies may reflect the source(yeast vs. bacterial), and hence the activity, of the protein used.

While wt-NK1 remains less active than full length HGF/SF, remarkably thetwo K domain mutants exhibited biological activity much higher thanwt-NK1 and equal or higher to full length HGF/SF. Our biochemical datasuggest that the K domain mutations result in increased net affinity ofNK1 for heparin. Thus the patch of amino acids consisting of K132, R134and R181 acts as a negative effector of heparin binding to NK1 andreverse-charge mutations of these residues increases heparin bindingprobably via the main site in the N domain.

Regardless of the mechanism, we have demonstrated that substitution oftwo amino acids in the K domain (K132:R134 or K170:R181) is sufficientfor converting NK1 into a full receptor agonist. NK1, but not HGF/SF,can be produced in yeast and is expected to exhibit favourable in vivokinetics and tissue distribution compared to full length HGF/SF.

EXAMPLE 1

In this example the production and analysis of the biological activityof two NK1 variants of the invention is illustrated. We show that thesevariants undergo dimerization in a manner similar to NK1 in the presenceof heparin and are rendered potent receptor agonists through mutagenesisof a cluster of positively-charged residues on the K domain.

Materials and Methods

Cloning, mutagenesis, expression and purification Cloning, expressionand purification of wt-NK1 were carried out as described in (Chirgadzeet al., 1999), except that final purification of NK1 by cation exchangechromatography was carried out on a Source15S column (Amersham PharmaciaBiotech). For expression of NK1 mutants, an EcOR1-NotI fragment from thewt-NK1 expression construct in pPIC9K was cloned into the pBluescriptKS-vector (Stratagene) and DNA amplification reactions were carried outusing complementary pairs of mutagenic oligonucleotides. The N-domainmutants were produced by DNA amplification of the relevant fragmentsfrom full length human HGF/SF cDNA which carried R73E:R76E mutations(mutant HP11) or the (K58E:K60E:K62E) mutations (mutant HP12). N domainand K domain mutants were finally cloned in the expression vectorpPIC9K. Transformation and selection of P. pastoris was carried out asdescribed previously (Chirgadze et al., 1999).

Purification of Heparin Fragments

Sodium heparin from bovine lung (Upjohn) was digested with heparinase I(Leo Pharmaceuticals) for 14 min at 37° C. in 10 mM phosphate buffer,1.25 mM CaCl₂, pH 7.0. The water was evaporated, the residue dissolvedin 20 g/l ammonium bicarbonate and loaded onto a Biogel P-10 (Biorad)column. Fractions containing heparin fragments of the same length (up tohexadecasaccharide) were combined and water and ammonium bicarbonateevaporated on a Rotavapor (Buechi). The heparin fragments were thendissolved in 0.1 M ammonium acetate and an aliquot was run through aG3000 SW XL (30 cm×7.8 mm) and a G2000 SW XL (30 cm×7.8 mm) GPC columnon a HPLC system (Gilson) in order to assess purity and concentration.The fragments were next lyophilized and redissolved in water (3 cycles)in order to eliminate ammonium acetate.

Characterization of wt- and Mutant NK1-Heparin Complexes

This was carried out by gel filtration chromatography and cross-linkingexperiments. For gel filtration, wt- or mutant NK1 (0.5 mg/ml) wereincubated for 2 hours in the presence or absence of equimolarconcentration of 14-mer heparin in phosphate buffered saline (PBS)adjusted to 300 mM NaCl. Samples were then loaded onto an HR30 Superdex200 column (Ammersham Pharmacia Biotech) and eluted at 0.5 ml/min.

For cross-linking, 10 μl of wt- or mutant-NK1 (0.1 mg/ml) were incubatedin the absence or presence of an equimolar concentration of 14-merheparin in PBS. After 2 hours incubation at room temperature, 1 μl ofcrosslinker (BS³, Pierce) was added at 100 fold molar excess, thereaction was continued for 30 minutes and then quenched with 1 μl of 1MTris-Cl, pH 7.4. Reaction products were loaded onto 15%SDS-polyacrylamide gels and blotted onto a nitrocellulose membrane(Schleicher & Schuell). The membrane was blocked in 2% skimmed milk,incubated for 1 hour in the presence of sheep anti-HGF/SF polyclonalantibody (1W53, 1:1000), washed with PBS+0.2% Tween 20 and nextincubated for 1 hour with HRP-conjugated anti-sheep immunoglobulinantibody (Dako). HRP activity was detected after 3 further washes inPBS+0.2% Tween 20 using a chemiluminescent substrate (Pierce).

MDCK Colony Scatter Assays

Scatter assays were carried out as described in (Gherardi et al., 1989;Stoker et al., 1987). Briefly, MDCK cells were plated at 1–2.5×10³cells/60 mm dish and cultured in 5% fetal calf serum in DMEM for 2–3days before addition of HGF/SF or wt- or mutant NK1. After overnightincubation, plates were inspected and several colonies from each platewere photographed using a Leica DM IRB inverted microscope equipped withphase contrast optics and a Hamamatsu colour chilled 3CCD camera.

DNA Synthesis in MK Cells

The mouse keratinocyte line MK was cultured to confluence inkeratinocyte SFM medium (Gibco) supplemented with 5 ng/ml EGF-53 and 50g/ml bovine pituitary extract (BPE) in 24 well tissue culture plates(Costar). At confluence, complete medium was replaced with basal medium(no EGF and BPE) for 24 h before addition of 1 Ci/well ³H-thymidine inbasal medium containing 1 mg/ml BSA and HGF/SF or NK1 proteins at theconcentrations specified in the legend to FIG. 1. After 16 hours thecells were transferred on ice, washed with PBS and incubated inice-cold, 5% trichloroacetic acid (TCA) for 30 min. TCA insolubleradioactivity was measured by scintillation after 2 washes with waterand lysis in 0.2 M NaOH for 30 minutes at 37C.

Results

The NK1 fragment of HGF/SF (amino acids 28–210) was expressed in themethylotrophic yeast P. pastoris as described (Chirgadze et al., 1999)and crystallized in complex with a tetrahexameric (14-mer) heparinfragment. The heparin fragment was prepared by digestion andpurification from polydisperse heparin extracted from bovine lung.

The crystallization of the protein in complex with heparin is describedin GB0110430.6 of 27 Apr. 2001, from which priority is hereby claimedand the contents of which are hereby incorporated by reference, and inLietha, D. et al.; EMBO J. 2001 Oct. 15;20(20):5543–55, whose contentsare also herby incorporated by reference. The crystallization andanalysis of the complex allowed the present inventors to identify aminoacid residues in NK1 which could be altered. Having identified suchresidues, those of skill in the art to produce variants of the inventionbased on the present disclosure herein.

Briefly, two crystal types were found. The asymmetric unit ofcrystal-type A contains two NK1 protomers, A and B, assembled into ahead-to-tail dimer, as in the previously described crystal structures ofNK1 (Chirgadze et al., 1999; Ultsch et al., 1998). A hepes molecule isbound to each of the K domains in the putative lysine-binding pocket, asin the lbht structure (Ultsch et al., 1998). A heparin molecule (H) wasclearly seen bound to the N domain of protomer A but the N domain ofprotomer B is partially disordered and poorly defined at the periphery.Thus it could not be seen whether a heparin molecule was bound. Theheparin molecule bound to protomer A also makes contacts with thekringle domain of protomer A′ from the neighbouring asymmetric unit ofthe crystals. The final refined structure contained 5 heparin sugarunits: 2 glycosamines (GlcN) and 3 iduronic acids (IdoA) of the 14present in the complex.

In contrast, the asymmetric unit of crystal type B contained an assemblyof four NK1 dimers (A & B, C & D, E & F, G & H) with six bound heparinmolecules. The dimers in the asymmetric unit were positioned in a circlewith a pseudo-two fold axis running through the centre. The NK1 dimerarrangement was identical to that observed in crystal type A anddescribed earlier (Chirgadze et al., 1999; Ultsch et al., 1998). Unlikethe structure of crystal type A, all residues between 38 and 208 arewell ordered and show clear electron density in all protomets. All Ndomains interact with heparin molecules. The N domains of protomers Aand E share a heparin molecule as do N domains of protomers D and G. Thelongest heparin fragment, that could be built into electron densitymaps, is nine sugar units in length; it is bound to the N domain ofprotomer C and the K domain of protomer F. Other heparin molecules areless defined with the shortest fragment containing only five sugar units(heparin molecule N). Each K domain has a hepes molecule bound in thesame binding pocket as in the structure of crystal type A. The dimerswithin the asymmetric unit show a good agreement with r.m.s.d. values ofCα atoms between 0.50 Å (comparing dimer consisting of protomers A and Bwith that consisting of protomers G and H) and 1.32 Å (comparing dimerconsisting of protomers A and B with that consisting of protomer C andD). The NK1 dimers in crystal type B are also very similar to the dimerin crystal type A, with the worst r.m.s.d. of Cα atoms amounting to 1.19Å for the dimer consisting of protomers A and B.

Heparin—K domain interactions were seen in both crystal structures andinvolved a cluster of positively charged residues (K132, R134, K170,R181). These residues form a patch of positive electrostatic potentiallining against the negatively charged heparin chain. The functionalsignificance of the heparin—K domain interactions was probed bymutagenesis.

Novel NK1 Mutants

Two reverse-charge N domain mutants (HP11 and HP12) and two K domainmutants (1K1 and 1K2) were generated (Table 1) and characterized forheparin binding and biological activity.

TABLE 1 NK1 Mutant Substitutions HP11 R73E: R76E HP12 K58E: K60E: K62E1K1 K132E: R134E 1K2 K170E: R181E

Cross-linking (Schwall et al., 1996) and gel filtration (Chirgadze etal., 1999) experiments were employed first in order to characterizeheparin-mediated oligomerization of wt NK1 in solution. Wild-type andmutant NK1 were incubated in the absence or presence of equimolarconcentrations of 14-mer heparin. Cross-linked proteins were analyzed bywestern blotting and detected with an anti-HGF/SF polyclonal antibody(1W53). In addition, gel filtration of wild type and mutant NK1 in theabsence or presence of equimolar concentrations of 14-mer heparin wasalso performed. Chromatography was carried out on an HR30 Superdex-200column equilibrated in PBS adjusted to 300 mm NaCl. Wt-NK1 and thedifferent mutants showed slight variations in elution volume due toresidual interaction with the column.

Heparin failed to induce both cross-linking and oligomerization insolution of the HP11 mutant. HP12, the second N domain mutant wascross-linked by heparin but, like the HP11 mutant, failed to oligomerizein solution in the presence of heparin. The two K domain mutants (1K1and 1K2), however, behaved like wt-NK1 in these experiments. Inconclusion, the amino acids that make crystallographic contact withheparin in the N domain are required for heparin-dependent dimerizationof NK1 in solution indicating that these amino acids are responsible forheparan sulphate-dependent dimerization of NK1 on the cell surface.

Biological Activity of NK1 Mutants

Experiments with heparan sulphate-deficient cells have established anessential requirement for heparan sulphate or soluble heparin for thebiological activity of NK1 (Schwall et al., 1996). Normal cells displaymembrane-bound heparan sulphate and thus, if they express the METreceptor, respond to NK1. They may fail, however, to respond toheparan-sulphate-deficient NK1 mutants such as HP11 and HP12.

Colony dispersion (scatter) assays with MDCK cells were performedessentially as described by Gherardi et al., 1989 and Stoker et al.,1987 in the presence of full length HGF/SF or NK1 proteins. Briefly,MDCK cells were plated at low density in 60 mm dishes and cultured for 3days in standard medium after which the medium was replaced with freshmedium or medium containing 10⁻¹⁰ M HGF/SF or 10⁻⁸ M of the various NK1proteins. After overnight incubation several colonies from each dishwere photographed using phase contrast optics.

The colonies in control cultures exhibited strong cell-cell adhesion anda typical epithelial, ‘cobblestone’ appearance. HGF/SF (10⁻¹⁰ M), wt-NK1or the K domain mutant 1K1 (10⁻⁸ M) induced full dissociation of MDCKcolonies. In contrast, both the HP11 and HP12 mutants (10⁻⁸ M) wereinactive. Addition of soluble heparin (10⁻⁶ M) did not affect controlcultures or cultures containing HGF/SF or NK1 proteins.

The biological activity of the NK1 mutants was studied further on adifferent target, the MK mouse keratinocyte line, which exhibits astrong mitogenic response to HGF/SF (Moorby et al., 1995). Wt-NK1, butnot the N domain mutants HP11 and HP12, induced appreciable stimulationof DNA synthesis at concentrations of 10⁻¹⁰ M or higher (FIG. 1).Remarkably, the K domain mutant 1K1 exhibited activity much higher thanwt-NK1 and comparable or even higher than that of full length HGF/SF.1K2, the second K domain mutant, behaved like and gave a similar resultas the 1K1 mutant. Thus, the HP11 and HP12 mutations that failed toinduce dimerization of NK1 in solution, also caused loss of biologicalactivity, presumably due to the inability of these mutants to bindcell-associated heparan sulphate on the surface of MDCK or MK cells. Incontrast, the K domain mutations conferred increased biological activityto NK1 and converted it to a full receptor agonist.

In order to establish whether the loss of activity of the HP11 and HP12mutants was due to defective receptor binding or activation, competitionexperiments were carried out in which MDCK cells were cultured in thepresence of HGF/SF alone (10⁻¹⁰ M) or in the presence of HGF/SF andexcess concentrations (10⁻⁸ or 10⁻⁷ M) of wt-NK1 or the two N domainmutants. As expected, wt-NK1 behaved as a partial antagonist but HP11and HP12 exhibited no (HP11) or very little (HP12) antagonistic activityimplying that the lack of activity of these mutants is due to reducedreceptor binding rather than failure to induce receptor activation.

EXAMPLE 2 In Vivo Activity of 1K1

Three groups of 12 Balb/c male mice (10 weeks old, about 35 g) plus acontrol group of 20 such animals were administered 0.6 g/kg i.p. ofN-acetyl-p-aminophenol in 0.3 ml PBS. Following dosing, mice weretreated at two hours and six hours with 0.5 mg/kg i.v. of 1K1, NK1 orHGF/SF or, in the case of the control group, left untreated.

The results are shown in FIG. 2. Briefly, N-acetyl-p-aminophenol causeddeath in 85% of the animals that received drug but no growth factor overa period of 3 days, with just under 50% of the animals dead 4 hoursafter treatment. HGF/SF offered some protection and, somewhatsurprisingly, NK1 was more active than HGF/SF, achieving a 40% survival3 days after treatment. The NK1 mutant 1K1 was the most effective of theprotein tested resulting in 80% survival.

EXAMPLE 3 Activity of NK1 and 1K1 in -Amanitin-induced Liver Failure

Three groups of 12 test mice and a control group of 20 mice of the samestrain, size and sex as Example 2 were administered by i.p. 0.9 mg/kgα-amanitin. The test groups were then given 5 injections every 12 hours,commencing 12 hours after α-amanitin dosing, of 0.5 mg/kg i.v. of NK1,1K1 or HGF/SF. The results are shown in FIG. 3, indicating that both 1K1and NK1 were effective in reducing early stage (3–5 days) hepatictoxicity.

REFERENCES

-   Bladt, F., et al (1995). Essential role for the c-met receptor in    the migration of myogenic precursor cells into the limb bud [see    comments], Nature 376, 768–71.-   Bottaro, D. P., et al (1991). Identification of the hepatocyte    growth factor receptor as the c-met proto-oncogene product, Science    251, 802–4.-   Caton, A., et al (2000). The branchial arches and HGF are    growth-promoting and chemoattractant for cranial motor axons,    Development 127, 1751–66.-   Chan, A. M., et al (1991). Identification of a competitive HGF    antagonist encoded by an alternative transcript, Science 254,    1382–5.-   Chirgadze, D. Y., et al (1999). Crystal structure of the NK1    fragment of HGF/SF suggests a novel mode for growth factor    dimerization and receptor binding, Nat Struct Biol 6, 72–9.-   Cioce, V., et al (1996). Hepatocyte growth factor (HGF)/NK1 is a    naturally occurring HGF/scatter factor variant with partial    agonist/antagonist activity, J Biol Chem 271, 13110–5.-   Donate, L. E., et al (1994). Molecular evolution and domain    structure of plasminogen-related growth factors (HGF/SF and    HGF1/MSP), Protein Sci 3, 2378–94.-   Ebens, A., et al (1996). Hepatocyte growth factor/scatter factor is    an axonal chemoattractant and a neurotrophic factor for spinal motor    neurons, Neuron 17, 1157–72.-   Gherardi, E., et al (1989). Purification of scatter factor, a    fibroblast-derived basic protein that modulates epithelial    interactions and movement, Proc Natl Acad Sci USA 86, 5844–8.-   Hartmann, G., et al (1992). A functional domain in the heavy chain    of scatter factor/hepatocyte growth factor binds the c-Met receptor    and induces cell dissociation but not mitogenesis, Proc Natl Acad    Sci USA 89, 11574–8.-   Hartmann, G., et al (1998). Engineered mutants of HGF/SF with    reduced binding to heparan sulphate proteoglycans, decreased    clearance and enhanced activity in vivo. Curr Biol 8, 125–34.-   Jakubczak, J. L., et al (1998). NK1, a natural splice variant of    hepatocyte growth factor/scatter factor, is a partial agonist in    vivo, Mol Cell Biol 18, 1275–83.-   Jeffers, M., et al (1996). Hepatocyte growth factor/scatter    factor-Met signaling in tumorigenicity and invasion/metastasis, J    Mol Med 74, 505–13.-   Lokker, N. A., and Godowski, P. J. (1993). Generation and    characterization of a competitive antagonist of human hepatocyte    growth factor, HGF/NK1, J Biol Chem 268, 17145–50.-   Miyazawa, K., et al (1991). An alternatively processed mRNA    generated from human hepatocyte growth factor gene, Eur J Biochem    197, 15–22.-   Miyazawa, K., et al (1989). Molecular cloning and sequence analysis    of cDNA for human hepatocyte growth factor, Biochem Biophys Res    Commun 163, 967–73.-   Mizuno, K., et al (1994). Hairpin loop and second kringle domain are    essential sites for heparin binding and biological activity of    hepatocyte growth factor, J Biol Chem 269, 1131–6.-   Moorby, C. D., et al. (1995). HGF/SF inhibits junctional    communication, Exp Cell Res 219, 657–63.-   Nakamura, T., et al (1989). Molecular cloning and expression of    human hepatocyte growth factor, Nature 342, 440–3.-   Rapraeger, A. C., et al (1991). Requirement of heparan sulfate for    bFGF-mediated fibroblast growth and myoblast differentiation,    Science 252, 1705–8.-   Schmidt, C., et al (1995). Scatter factor/hepatocyte growth factor    is essential for liver development, Nature 373, 699–702.-   Schmidt, L., et al (1997). Germline and somatic mutations in the    tyrosine kinase domain of the MET proto-oncogene in papillary renal    carcinomas, Nat Genet 16, 68–73.-   Schwall, R. H., et al (1996). Heparin induces dimerization and    confers proliferative activity onto the hepatocyte growth factor    antagonists NK1 and NK2, J Cell Biol 133, 709–18.-   Stoker, M., et al (1987). Scatter factor is a fibroblast-derived    modulator of epithelial cell mobility, Nature 327, 239–42.-   Uehara, Y., et al (1995). Placental defect and embryonic lethality    in mice lacking hepatocyte growth factor/scatter factor, Nature 373,    702–5.-   Ultsch, M., et al (1998) Crystal structure of the NK1 fragment of    human hepatocyte growth factor at 2.0 A resolution, Structure 6,    1383–93.-   Yayon, A., et al (1991). Cell surface, heparin-like molecules are    required for binding of basic fibroblast growth factor to its high    affinity receptor, Cell 64, 841–8.-   Yu, J., Miehlke, S., et al (2000). Frequency of TPR-MET    rearrangement in patients with gastric carcinoma and in first-degree    relatives, Cancer 88, 1801–6.-   Zhou, H., et al (1999). Identification and dynamics of a    heparin-binding site in hepatocyte growth factor, Biochemistry 38,    14793–802.-   Zhou, H., et al (1998). The solution structure of the N-terminal    domain of hepatocyte growth factor reveals a potential    heparin-binding site, Structure 6, 109–16.

1. A polypeptide comprising SEQ ID NO:1, wherein the amino acid of atleast one of positions 105, 107, 143 or 154 is substituted.
 2. Thepolypeptide of claim 1, wherein the amino acids at positions 105 and 107are substituted.
 3. The polypeptide of claim 2, wherein saidsubstitutions are K105E and R107E.
 4. The polypeptide of claim 1,wherein the amino acids at positions 143 and 154 are substituted.
 5. Thepolypeptide of claim 4, wherein said substitutions are K143E and R154E.6. A polypeptide comprising a fragment of SEQ ID NO:2, comprising aminoacids 132–181, wherein the amino acid of at least one of positions 132,134, 170 or 181 is substituted, and said fragment exhibitsheparin-dependent dimerization and is a MET receptor agonist.
 7. Apolypeptide comprising a fragment of SEQ ID NO:2, comprising amino acids32–206, wherein the amino acid of at least one of positions 132, 134,170 or 181 is substituted, and said fragment exhibits heparin-dependentdimerization and is a MET receptor agonist.
 8. A polypeptide comprisinga fragment of SEQ ID NO:2, comprising amino acids 128–206, wherein theamino acid of at least one of positions 132, 134, 170 or 181 issubstituted, and said fragment exhibits heparin-dependent dimerizationand is a MET receptor agonist.
 9. A composition comprising thepolypeptide of any one of claims 1–8, and a pharmaceutically acceptablediluent or carrier.